Hirudin is a polypeptide with anti-thrombotic activity which is isolated from the salivary glands of the medicinal leech, Hirudo medicinalis. Three hirudin analogs, HV1, HV2 and HV3 are known as natural hirudins. The amino acid sequence of these natural hirudins has been identified. The genes coding the identified amino acid sequence been cloned as c-DNAs, and the DNA sequences have also been identified.
Expression vectors were prepared using the c-DNAs cloned from natural genes. Expression of the polypeptides, the hirudin analogs, has been carried out by introducing the expression vectors into host microorganisms such as E. coli (Escherichia coli) and yeast (Saccharomyces cerevisiae).
Furthermore, based on knowledge related to the amino acid sequences of the natural hirudins, hirudin analogs with an artificial mutation of the amino acid sequence have been produced. The following are reported as examples. HV2(Lys.sup.47) was made by introducing one amino acid substitution into the natural hirudin HV2. This hirudin analog exhibited an increased antithrombin activity as compared with that of the natural hirudin HV2, and exhibited a high antithrombin activity equivalent to that of the natural hirudin HV1. Other examples include an analog of the natural hirudin HV1 in which the amino acid sequence of the N-terminal region was substituted with other amino acids, and the HV1-like analog having extra amino acids attached to its N-terminal. An analog made by replacing the amino acid sequence of the N-terminal of natural hirudin HV1 from Val.sup.1 -Val.sup.2 - to Ile.sup.2 -Ile.sup.2 - was reported to exhibit a higher antithrombin activity than that of the natural hirudin HV1. The N-terminal region of a hirudin is thought to form an intermolecular bond with the enzyme active center or its adjacent region in the thrombin polypeptide chain. This region is also thought to control the dissociation rate of the hirudin-thrombin complex.
In contrast to the idea of making hirudin analogs by introducing artificial point mutations to the natural type hirudins as mentioned above, the present inventors have produced hybrid hirudins such as HV1C3 by substituting the C-terminal region polypeptide of the HV1 with an C-terminal region polypeptide of the HV3. Among these, the present inventors have demonstrated that this hybrid type hirudin HV1C3 shows a higher antithrombin activity than that of the hirudin HV1 preferably insert EP-A-511393 (Japanese laid-open patent publication 4-173798). Furthermore, such hirudins exhibiting high antithrombin activities were shown to have pharmacological efficacy as antithrombotics which suppress the coagulation process caused by the digestion of polypeptide chains by thrombin. In other words, it has been clearly demonstrated that the coagulation inhibition or suppression properties of the compounds were due to their antithrombin activity.
The present inventors have prepared anticoagulants with the above-mentioned hybrid hirudin analogs such as HV1C3, which was proposed in our previous patent application, as active ingredients in such a manner so that one pure hirudin analog is contained in a certain pharmacological equivalent amount. When these formulations were stored at room temperature for an extended period, changes in the apparent pharmacological activities were observed. The present inventors have investigated the causes of these changes and have discovered the fact that, in the case of HV1C3, a succinimide form and a .beta. form of the Asp.sup.33 -Gly.sup.34 were formed between the carboxyl residue of the Asp.sup.33 and its C-terminal adjacent to Gly.sup.34 (Japanese Laid-Open Patent Publication No. 5-310788). This succinimide form and .beta. form exhibit antithrombin activities like the original hirudin, but the activities are lower, which lead to a decrease in the apparent antithrombotic activities. Furthermore, based on a report that a succinimide form or a .beta. form can be produced by a chemical reaction between the Asp and the amino acid adjacent to it on the C-terminal side, the present inventors have studied this chemical conversion in more detail and have found a possibility that the succinimide form or .beta. form can also be produced at Asp.sup.62 -Ala63-.
This succinimide form and .beta. form (hereafter "hirudin variants") can be produced during the storage following preparation of a hirudin anticoagulant in an appropriate formulation, and this requires extra considerations to keep the pharmacological activity of the anticoagulant agent above a certain preferred level of activity. These might include a low temperature storage, for example, or an increase in the initial amount of hirudin analogs in the formulation in consideration of loss of the pharmacological activity during room temperature storage. Increasing the initial amount of hirudin analogs in the formulation, however, causes problems by unnecessarily elongating the clearance time of the drug in the blood following administration.